Early stages of wing differentiation. (A) To illustrate the morphogenetic process of wing-disc elongation, wing tissue from late third larval instar (L3), 2 h after puparium formation (APF), and 36 h APF was dissected and stained for DNA. The wing margin (red), wing veins (orange), and notum (purple) are indicated in developing tissue and the adult fly. (B) Between L3 and 36 h APF, each wing epithelial cells adopts a cell-type-specific shape, and differentiates a wing hair. Time courses of wings stained for either DE-cadherin (to visualize apical cell shape) or F-actin (to visualize hair formation) are shown. Images are centered on a presumptive wing vein. Developmental stages are indicated. (C) Flow cytometric analysis demonstrates changes in DNA content associated with cell-cycle exit in the wing. At L3 and 2 h APF, presumptive wing cells asynchronously proliferate. By 6 h APF, most cells in the wing temporarily arrest in the G2 phase of the cell cycle, leading to a relatively synchronized final cell cycle between 14 and 24 h APF (represented here by 18 h APF). By 24 h APF, cell proliferation is no longer detected in the wing epithelium, and nearly all cells arrest with a G1 DNA content. Dissected wing tissues stained for DNA are shown for each developmental stage. (D) To determine the changes in gene expression associated with wing morphogenesis and cell cycle exit, RNA was collected from six developmental time points between L3 and 36 h APF (corresponding to images shown in (C)), and microarray analysis was performed. Using L3 as a reference sample, the number of transcripts that exhibit a significantly different level of expression is listed for each time point.